The Transient Receptor Potential (TRP) proteins are a family of ion channels which are divided into three major subfamilies: The TRPC “Canonical”, the TRPV “Vanilloid”, and the TRPM “Melastatin” (see Clapham D E. Nature, 426, 517-24 (2003) Harteneck C et al. Trends Neurosci, 23, 159-66.(2000), Montell C, et al. Mol Cell, 9, 229-31(2002)). The TRPM subfamily consists of eight members and information regarding their physiological function has just begun to surface. TRPM4 is a widely expressed calciumactivated non-selective cation (CAN) channel that conducts mainly Na+ and K+ without appreciable permeation to Ca2+. It has a single channel conductance of ˜25 pS and is directly activated by [Ca2+]i. Two splice variants have been described, a short form, which lacks 174 amino acid residues at the N-terminus (Xu XZ, et al. Proc Natl Acad Sci U S A, 98, 10692-7 (2001)) and a long (full-length) form (Launay P, et al. Cell, 109, 397-407 (2002)). In non-excitable cells such as T-lymphocytes, the TRPM4-mediated depolarization reduces the driving force for Ca2+ entry through Ca2+ Release-Activated Ca2+ channels (CRAC) with significant impact on Ca2+ oscillations and cytokine production (Launay P, et al. Science, 306, 1374-7 (2004)). TRPM4 is also implicated in myogenic constriction and cardiac function (Earley S, et al. Circ Res, 95, 922-9 (2004); Guinamard R, et al. Physiol, 558, 75-83 (2004)), suggesting that it may critically regulate Ca2+ entry mechanisms in electrically excitable cells as well.
Changes in membrane potential during glucose stimulation are crucial for determining the shape and frequency of Ca2+ oscillations in β-cells, because each depolarization induces a concomitant rise in the [Ca2+]i, that triggers insulin secretion (Bergsten P. Diabetes, 51 Suppl 1, S171-6 (2002); Gilon P, et al. Diabetes, 51 Suppl 1, S144-51 (2002)) Impaired Ca2+ oscillations result in deficiencies in insulin secretion in certain forms of type 2 diabetes in humans and rodents (Henquin J C. Diabetes, 49, 1751-60 (2000); Lin J M, et al. Diabetes, 51, 988-93 (2002); O'Rahilly S, et al. N Engl J Med 318, 1225-30 (1988). The cellular and molecular components involved in membrane depolarization of β-cells have not been fully identified. Glucose stimulates insulin secretion by activating two pathways (Henquin J C. (2000). The triggering pathway involves a sequence of events beginning with glucose uptake, its metabolism and increase in ATP-ADP ratio, followed by closure of ATP-sensitive K+ (KATP) channels. Closure of KATP channels triggers membrane depolarization with opening of voltage-dependent calcium channels (VDCC's) and Ca2+ influx (Ashcroft F M, et al. Nature, 312, 446-8 (1984)), however, this requires the additional presence of a depolarizing current that so far has not been identified. The opening of VDCC's is dependent on the cell membrane potential, which is around −70 mV at rest. Depolarization activates VDCC's, with peak Ca2+ currents around 0 mV (Barg S, et al. Diabetes, 51 Suppl 1, S74-82 (2002); Berggren P O, et al. Cell, 119, 273-84 (2004); Gopel S, et al. J Physiol, 521 Pt 3, 717-28 (1999). TRPM4 currents reverse around 0 mV, and enhanced channel activity depolarizes cells from negative resting membrane potentials (launay P, et al. Cell, 109, 397-407 (2002)). The amplifying pathway, also referred to as the KATP-independent pathway, depends on an already elevated [Ca2+]i. I acts by increasing the efficiency of Ca2+ on secretion.
The global diabetes epidemic has resulted in a need for agents that can treat the symptoms of this illness. Of crucial importance in controlling diabetes is the ability to control and modulate insulin levels in the blood. Accordingly, the present invention provides methods for screening for candidate agents which can modulate insulin secretion from insulin secreting cells.